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United States Patent |
5,637,569
|
Magnusson
,   et al.
|
June 10, 1997
|
Ganglioside analogs
Abstract
Ganglioside lactam analogue derivatives of general formula (I) in which A
is a sialic acid residue of formula (II) which is bound via the dashed
line in the 2-position; and in which Z.sup.1 is --OH or a group
--NHX.sup.1, and Y.sup.30 is --CH.sub.3 or --CH.sub.2 OH; X.sup.1 and
Y.sup.1 together form a bond and Y.sup.2 is --OH or a group OR.sup.20, or
X.sup.1 and Y.sup.2 together form a bond and Y.sup.1 is --OH or --NHAc;
Y.sup.3 is --OH or a group --OR.sup.20 ; R.sup.1 is H or a sialic acid
residue of formula (III) which is bound via the dashed line in the
2-position; and in which Z.sup.2 is --OH or a group --NHX.sup.2, and
Y.sup.30 is as defined above; X.sup.2 and Y.sup.10 together form a bond
and Y.sup.20 is --OH, or X.sup.2 and Y.sup.20 together form a bond and
Y.sup.10 is --OH; with the provisos that when R.sup.1 is H, then Z.sup.1
is --NHX.sup.1, and that when R.sup.1 is a sialic acid residue of formula
(III), then at least one of Z.sup.1 and Z.sup.2 is different from --OH;
R.sup.10 is H, a carrier CA, or a group -(Sugar).sub.n. The compounds are
hydrolytically stable lactam analogs which spatially closely resemble the
naturally occurring ganglioside lactones and are antigenic and able to
induce the production of antibodies that cross-react with the
corresponding ganglioside lactone.
Inventors:
|
Magnusson; Hans G. (Lund, SE);
Ray; Asim K. (Lund, SE)
|
Assignee:
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Symbicom Aktiebolag (Umea, SE)
|
Appl. No.:
|
232240 |
Filed:
|
September 12, 1994 |
PCT Filed:
|
November 11, 1992
|
PCT NO:
|
PCT/DK92/00333
|
371 Date:
|
September 12, 1994
|
102(e) Date:
|
September 12, 1994
|
PCT PUB.NO.:
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WO93/10134 |
PCT PUB. Date:
|
May 27, 1993 |
Foreign Application Priority Data
| Nov 11, 1991[DK] | 1853/91 |
| Jul 03, 1992[DK] | 0880/92 |
Current U.S. Class: |
514/25; 536/4.1; 536/17.2; 536/17.9; 536/18.7; 536/55.2; 536/55.3 |
Intern'l Class: |
A61K 031/70; C07H 015/10 |
Field of Search: |
536/4.1,18.7,53,55.2,53.3,17.2,17.9
574/25
|
References Cited
Foreign Patent Documents |
0167449 | Jan., 1986 | EP.
| |
0315113 | May., 1989 | EP.
| |
0319253 | Jun., 1989 | EP.
| |
Other References
Nores et al., Density-Dependent Recognition of Cell Surface GM.sub.3 by a
Certain Anti-Melanoma Anti-Melanoma Antibody, and GM.sub.3 Lactone as a
Possible Immunogen; Requirements for Tumor-Associated Antigen and
Immunogen, The Journal of Immunology, vol. 139, No. 9, pgs. 3171-3176,
Nov. 1, 1987.
Ray et al., Synthesis nad conformational Analysis of GM.sub.3 Lactam, a
Hydrolytically Stable Analogue of GM.sub.3 Ganglioside Lactone, J. AM.
Chem. Soc., vol. 114, pp. 2256-2257, 1992.
|
Primary Examiner: Peselev; Elli
Attorney, Agent or Firm: Cooper; Iver P.
Claims
We claim:
1. A compound which is a ganglioside lactam analogue derivative of the
formula I
##STR13##
in which A is a sialic acid residue of the formula II
##STR14##
which is bound via the dashed line in the 2-position; and in which
Z.sup.1 is --OH or a group --NHX.sup.l, and Y.sup.30 is --CH.sub.3 or
--CH.sup.2 OH;
when Z.sup.1 is --NHX.sup.1, X.sup.1 and Y.sup.1 together form a bond and
Y.sup.2 is --OH or a group OR.sup.2, or X.sup.1 and Y.sup.2 together form
a bond and Y.sup.1 is --OH or --NHAc;
Y.sup.3 is --OH or a group --OR.sup.20 ;
R.sup.1 is H or a sialic acid residue of the formula III
##STR15##
which is bound via the dashed line in the 2-position; and in which
Z.sup.2 is --OH or a group --NHX.sup.2, and Y.sup.30 is as defined above;
when Z.sup.2 is --NHX.sup.2, X.sup.2 and Y.sup.10 together form a bond and
Y.sup.20 is --OH, or X.sup.2 and Y.sup.20 together form a bond and
Y.sup.10 is --OH; with the provisos that when R.sup.1 is H, then Z.sup.1
is --NHX.sup.1, and that when R.sup.1 is a sialic acid residue of formula
III above, then at least one of Z.sup.1 and Z.sup.2 is different from
--OH;
R.sup.10 is H, a carrier, or a group-(Sugar).sub.n, in which Sugar is a
monosaccharide unit selected from the group consisting of D-glucose,
D-galactose, D-mannose, D-xylose, D-ribose, D-arabinose, L-fucose,
2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose,
D-glucuronic acid, D-galacturonic acid, D-mannuronic acid,
2-deoxy-2-phthalimido-D-glucose, 2-deoxy-2-phthalimido-D-galactose, and
sialic acid, and n is an integer from 1 to 10, and in which the
reducing-end terminal sugar unit is either a hemiacetal or is
glycosidically bound to a pharmaceutical or a carrier CA;
R.sup.20 is a group of the formula I'
##STR16##
in which m is an integer 0 or 1; p is an integer from 1 to 5; Sugar1 is a
monosaccharide unit selected from the group consisting of D-glucose,
D-galactose, D-mannose, D-xylose, D-ribose, D-arabinose, L-fucose,
2-acetaraido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose,
D-glucuronic acid, D-galacturonic acid, D-mannuronic acid,
2-deoxy-2-phthalimido-D-glucose, 2-deoxy-2-phthalimido-D-galactose, and
sialic acid; and
R.sup.3 is H or a sialic acid residue of the formula II defined above, in
which Z.sup.1, R.sup.l, Y.sup.10, Y.sup.20, Y.sup.30 and Z.sup.2 are as
defined above, and X.sup.1 and
Y.sup.1 together form a bond and Y.sup.21 is --OH, or X.sup.1 and Y.sup.21
together form, a bond and Y.sup.1 is --OH or --NHAc.
2. A compound as claimed in claim 1 in which at the most one of Y.sup.2 and
Y.sup.3 is a group --OR.sup.20.
3. A compound as claimed in claim 2 in which Y.sup.3 is --OH and each Sugar
unit in R.sup.10, if present, as well as the ring structure and the Sugar1
unit in the formula I', if present, are selected from the group consisting
of D-glucose, D-galactose, 2-acetamido-2-deoxy-D-galactose, and L-fucose
units.
4. A compound as claimed in claim 3 in which the ring in the general
formula I has the galacto configuration.
5. A compound as claimed in claim 4 in which R.sup.1 is H.
6. A pharmaceutical composition for the treatment of human cancer having
ganglioside lactones as cancer-associated antigens, which comprises a
compound according to claim 1 and a pharmaceutically acceptable excipient.
7. The compound of claim 1 in which R.sup.10 is a macromolecular carrier,
or a group -(Sugar).sub.n in which the reducing-end terminal sugar is
glycosidically bound to a macromolecular carrier.
Description
FIELD OF THE INVENTION
The present invention relates to analogs of gangliosides, a method for
preparation of such compounds, use of such compounds to induce immune
response and as inhibitors of adhesion of bacteria and viruses, as well as
in the production of antibodies against the compounds, antibodies against
the compounds as well as use of the antibodies in therapy or diagnosis.
BACKGROUND OF THE INVENTION
Gangliosides, i.e. sialic acid-containing glycosphingolipids, are
well-known components of the mammalian cell surfaces. Furthermore,
gangliosides have been found to occur in equilibrium with the
corresponding lactones. Thus, GM.sub.3 - and GD.sub.3 -ganglioside
[NeuAc.alpha.(2-3)Gal.beta.(1-4)Glc.beta.O-Ceramide and
NeuAc.alpha.(2-8)NeuAc.alpha.(2-3)Gal.beta.(1-4)Glc.beta.O-Ceramide] have
been demonstrated to form lactones where the sialic acid-derived carbonyl
group enters into lactone formation with various hydroxyl groups of the
NeuAc and Gal residues. Furthermore, it has been shown that such lactones
exist in vivo, e.g. in brain tissue and in the membranes of tumour cells,
and are not just artifacts of the isolation procedures (Gross, S. K.;
Williams, M. A.; McCluer, R. H. J. Neurochem. 1980, 34, 1351-1361). It has
also been shown that GM.sub.3 -lactone is much more immunogenic than is
the open form of GM.sub.3 -ganglioside (Nores, G. A.; Dohi, T.; Taniguchi,
M.; Hakomori, S.-I. J. Immun. 1987, 139, 3171-3176).
It has also been suggested that GM.sub.3 -lactone is a highly rigid
structure with the lactone ring in a chair-like conformation (see scheme 1
below) which would mean that its ability to serve as a recognition site or
membrane antigen could be enhanced, compared to its parent structure,
which is flexible (Yu, R. K.; Koerner, T. A. W.; Ando, S.; Yohe, H. C.;
Prestegaard, J. H. J. Biochem. 1985, 98, 1367-1373). Furthermore, the
lactone of GM.sub.3 -ganglioside ("GM.sub.3 -lactone") has been suggested
to be a tumour-associated antigen on the cells of an experimental mouse
melanoma (Nores, G. et al. cited above). In a comparative immunization
with GM.sub.3 -ganglioside and the corresponding lactone, the same authors
showed that the lactone is the stronger immunogen, and it was suggested
that this compound could be the real immunogen despite being a minor
membrane component. Furthermore, the activity of a monoclonal
anti-melanoma antibody (M2590) with various cells and liposomes was shown
to depend in a threshold, all-or-none, fashion on the concentration of
GM.sub.3 -ganglioside in the cell membrane or liposome. Also, the antibody
was found to cross-react with GM.sub.3 -lactone.
The ganglioside lactones are unstable at neutral pH while acidic conditions
favour the lactone in the equilibrium between GM.sub.3 -ganglioside and
GM.sub.3 -lactone. However, since GM.sub.3 -ganglioside is itself acidic
since it is an acid-containing glycolipid, the ganglioside might possibly
induce its own lactonization when the concentration is sufficiently high
in a cell membrane or liposome. This might help to explain the threshold
effect described above and might have similar implications for other
sialic acid-containing saccharides.
Thus, although a ganglioside lactone has been shown to be far more
immunogenic than the open-form ganglioside, the low equilibrium
concentration of the lactone at pH-values close to neutral will probably
render the lactone to be a rather inefficient immunogen. Thus, it is clear
that it would be desirable to have a non-labile immunogenic compound
resembling the lactone form of a ganglioside and which is capable of
raising antibodies (and other entities of the immune system) capable of at
least partial cross-reaction with the ganglioside lactone.
SUMMARY OF THE INVENTION
In one aspect, the present invention relates to ganglioside analogs which
spatially closely resemble gangliosides and which are therefore
contemplated to be able to induce the production of antibodies that will
cross-react with the corresponding ganglioside and in turn elicit an
immune response directed against the gangliosides and thereby against
entities on which the gangliosides are present.
The invention therefore relates to ganglioside lactam analogue derivatives
of the general formula I
##STR1##
in which A is a sialic acid residue of the formula II
##STR2##
which is bound via the dashed line in the 2-position; and in which
Z.sup.1 is --OH or a group --NHX.sup.1, and Y.sup.30 is --CH.sub.3 or
--CH.sub.2 OH;
X.sup.1 and Y.sup.1 together form a bond and Y.sup.2 is --OH or a group
OR.sup.20, or
X.sup.1 and Y.sup.2 together form a bond and Y.sup.1 is --OH or --NHAc;
Y.sup.3 is --OH or a group --OR.sup.20 ;
R.sup.1 is H or a sialic acid residue of the formula III
##STR3##
which is bound via the dashed line in the 2-position; and in which
Z.sup.2 is --OH or a group --NHX.sup.2, and Y.sup.30 is as defined above;
X.sup.2 and Y.sup.10 together form a bond and Y.sup.20 is --OH, or
X.sup.2 and Y.sup.20 together form a bond and Y.sup.10 is --OH; with the
provisos that when R.sup.1 is H, then Z.sup.1 is --NHX.sup.1, and that
when R.sup.1 is a sialic acid residue of formula III above, then at least
one of Z.sup.1 and Z.sup.2 is different from --OH; R.sup.10 is H, a
carrier CA, or a group -(Sugar).sub.n, in which Sugar is a monosaccharide
unit selected from the group consisting of D-glucose, D-galactose,
D-mannose, D-xylose, D-ribose, D-arabinose, L-fucose,
2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose,
D-glucuronic acid, D-galacturonic acid, D-mannuronic acid,
2-deoxy-2-phthalimido-D-glucose, 2-deoxy-2-phthalimido-D-galactose, and
sialic acid, and n is an integer from 0 to 10, and in which the
reducing-end terminal sugar unit is either a hemiacetal or is
glycosidically bound to a carrier CA;
R.sup.20 is a group of the formula I'
##STR4##
in which m is an integer 0 or 1; p is an integer from 1 to 5;
Sugar1 is a monosaccharide unit selected from the group consisting of
D-glucose, D-galactose, D-mannose, D-xylose, D-ribose, D-arabinose,
L-fucose, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose,
D-glucuronic acid, D-galacturonic acid, D-mannuronic acid,
2-deoxy-2-phthalimido-D-glucose, 2-deoxy-2-phthalimido-D-galactose, and
sialic acid; and
R.sup.3 is H or a sialic acid residue of the formula II defined above in
which Z.sup.1, R.sup.1, Y.sup.10, Y.sup.20, Y.sup.30 and Z.sup.2 are as
defined above, and X.sup.1 and Y.sup.1 together form a bond and Y.sup.21
is --OH, or X.sup.1 and Y.sup.21 together form a bond and Y.sup.1 is --OH
or --NHAc.
As it will be seen, the compounds of the present invention are lactam
analogs of corresponding ganglioside lactones, and such lactams are highly
stable at neutral pH and should provide a good lactone substitute. Thus,
in the case of GM.sub.3 -lactone it has been shown (as it will be
discussed below) that the corresponding GM.sub.3 -lactam has an extremely
similar over-all shape compared to the lactone which means that the lactam
is potentially able to induce the production of antibodies that will
cross-react with the corresponding ganglioside lactone and in turn elicit
an immune response directed against the ganglioside lactone and thereby
against entities on which the ganglioside lactone are present.
DETAILED DESCRIPTION OF THE INVENTION
As it will appear from the definition above, the group (Sugar).sub.n and
the group [Sugar1].sub.p is a group consisting of n and p monosaccharide
units, respectively, each of which are selected from those listed. It will
be apparent from formula I that the Sugar unit closest to the ring in
formula I is glycosidically linked (.alpha. or .beta.). Likewise, the
Sugar1 unit closest to the ring in formula I, i.e. the unit bound to the
6-position of the ring, is glycosidically linked (.alpha. or .beta.). The
entire group (Sugar).sub.n or [Sugar1].sub.p may be straight-chain or
branched, meaning that the aggregate of units in the entire group may form
a straight chain or a branched chain. Generally speaking, it is preferred
that each unit (apart from the reducing-end terminal unit) is bound to a
subsequent unit via a glycosidic linkage (.alpha. or .beta.). The
glycosidic linkage may connect to any of the available positions in the
subsequent unit, e.g. the 2-, 3-, 4- or 6-positions of the common
saccharide units or simple derivatives thereof or the 8-position of sialic
acid.
Furthermore, as also defined above, the reducing-end terminal saccharide
unit in the group (Sugar).sub.n may be either a hemiacetal, i,e. be
unsubstituted, or it may be glycosidically bound (.alpha. or .beta.) to a
carrier CA. Similarly, the ring in formula I may also be glycosidically
bound to a carrier CA, namely when R.sup.10 is such a carrier.
A carrier CA may be any of the aglycon groups that are found in natural or
synthetic glycoconjugates, glycosides (soluble or insoluble), glycolipids
or glycoproteins (as also discussed and exemplified in e.g. G. Magnusson
et al. J. Org. Chem. 1990, 55, 3932).
Thus, such an aglycon may be a lipid group, i.e. a lipophilic group which
together with the carbohydrate moiety forms a glycolipid. Examples of such
lipid groups are an acyl group of a fatty acid, e.g. a fatty acid with a
saturated or unsaturated, branched or unbranched hydrocarbon chain of 1-25
carbon atoms such as C.sub.1-20 alkanoic and alkenoic acids, an
aryl-containing group such as a benzoyl, phenyl or benzyl group, or
asteroid group such as a cholesterol or lanosterol group. Other examples
of lipid groups are a sphingolipid or glycerolipid group. The
glycosphingolipids or glycoglycerolipids arising as a result of the
coupling with the terminal Sugar unit have the following general
structures
##STR5##
Other lipid groups that can be coupled to the terminal Sugar unit may be
one of the aglycon groups described in U.S. Pat. No. 4,868,289 which is
hereby incorporated by reference. Such aglycon groups may preferably be
the sulphur-containing aglycons of the general formula
##STR6##
where q is an integer 0, 1, or 2, R.sub.4 is a saturated or unsaturated,
branched or unbranched alkyl chain of 1-25 carbon atoms, an aryl or
asteroid group, and R.sub.5 is H or a functional group such as CHO,
NO.sub.2, NH.sub.2, OH, SH, COOH, or CONH.sub.2. However, the functional
group, in particular COOH or CONH.sub.2, may further be bound to a large
or macromolecular structure as defined below.
The carrier CA may further be a large or macromolecular structure which may
be any organic or inorganic, polymeric or otherwise macromolecular
structure, examples of which are residues of proteins, polysaccharides,
plastic polymers and inorganic materials. Residues of proteins are
preferably bound through nucleophilic groups in the proteins, e.g. amino,
hydroxy, or mercapto groups. The proteins themselves may be any of a wide
variety of proteins, in particular biologically compatible proteins such
as globulins, albumins such as ovalbumin, bovine serum albumin (BSA) and
human serum albumin (HSA), fibrins, polylysin, "keyhole" limpet hemocyanin
(KLH), tetanus toxoid, etc. The polysaccharides may be any of a wide
variety of polysaccharides and may be bound through hydroxy groups on
ordinary polysaccharides such as cellulose, starch or glycogen, through
amino groups on aminosaccharides such as chitosane or aminated sepharose,
and through mercapto groups on thiomodified polysaccharides. Examples of
plastic polymers are aminated or thiolated latex, thiolated, aminated, or
hydroxylated polystyrene, and polyvinyl alcohol. Examples of inorganic
materials are aluminum oxide, or silicon oxide materials such as silica
gel, zeolite, diatomaceous earth, or the surface of various glass or
silica gel types such as thiolated or aminated glass, where the glass may
be in the form of e.g. beads.
Specific examples of carriers CA are illustrated below as they are attached
to the reducing-end terminal sugar unit.
##STR7##
Preferred compounds of the invention are those in which at the most one of
Y.sup.2 and Y.sup.3 is a group --OR.sup.20.
Among those compounds, particularly preferred compounds are those in which
Y.sup.3 is --OH and each Sugar unit in R.sup.10, if present, as well as
the ring structure and each Sugar1 unit in the formula I', if present, are
selected from D-glucose, D-galactose, 2-acetamido-2-deoxy-D-galactose, and
L-fucose units. In such compounds, it is especially preferred that the
ring in the general formula I has the galacto configuration. Also, it is
further preferred that R.sup.1 is H.
It is clear that for each of the two sialic acid residues of formula II and
III, there are two possible ways for the acid group on the sialic acid to
form a lactam with positions on the ring in formula I and in formula II,
respectively. Furthermore, a compound of the invention may, by virtue of
the definition of R.sup.3 in the group I', contain up to four sialic acid
residues with the consequent number of possible lactamization sites.
Furthermore, only at least one of the possible lactamization sites need
participate in a lactam function. This results in a considerable number of
possible specific structures for each basic ganglioside structure in terms
of the nature of the various carbohydrate functions [the ring in formulas
I and I', the nature of Sugar1 , and the nature and composition of
(Sugar).sub.n, as well as the remaining variables not involved in the
lactam formation]. To illustrate this relationship, the scheme below shows
the possible lactones (and the equilibrium reactions therebetween to which
the lactones, unlike the lactams of the invention, are subject to) that
can form from two gangliosides, namely (A) GM.sub.3 -ganglioside which
contains one sialic acid unit, and (B) GD.sub.3 -ganglioside which
contains two sialic acid units. The nomenclature used in the scheme and in
the rest of the present application is derived from the standard
nomenclature for the identification and characterization of gangliosides
that is described in the literature, in particular Methods in Enzymology,
vol. 179, part F, V. Ginsburg Ed., Academic Press Inc.
##STR8##
Examples of specific lactam structures are shown below where each compound
is named based on the ganglioside from which it is derived and with the
lactam functions indicated via the positions between which the lactam
bridge is formed.
##STR9##
Generally speaking, the compounds of formula I according to the invention
may, apart from the novel formation of the lactam rings, be prepared by
standard and well-established reaction sequences and procedures for
protection/deprotection and glycoside synthesis used in the preparation of
oligosaccharide glycoconjugates described in the literature such as by G.
Magnusson et al cited above. Lactam ring formation is usually performed
after completion of the construction of the oligosaccharide chain, after
which the aglycon carrier CA can be introduced. The nitrogen(s) that
is/are to be part of the lactam ring(s) may be introduced in protected
form at the monosaccharide level in the synthetic pathway and is then
deprotected and activated for ring-closure with the carboxylic moiety of
the sialic acid residue. Another and alternative route for the formation
of lactam rings is based on employing peptidase-induced lactamization of
NeuAc.alpha.2-3GalNAc residues where the N-acetyl group of GalNAc is
removed by means of the peptidase enzyme, thus leading to ring-closure of
the resulting free amino group with the carboxylic group of the NeuAc
residue.
More specifically, the glycosylation reactions may be carried out in an
aprotic, polar or non-polar organic solvent such as methylene chloride,
toluene, acetonitrile, ether, or nitromethane. The reaction temperature is
not critical and may range from -78.degree. C. to +150.degree. C.,
normally from 0.degree. C. to 50.degree. C. such as room temperature,
although yield maximization may be obtained through temperature
regulation. The reaction time may be from 0.1 to 200 hours, normally 1-24
hours such as 16 hours. The glycosyl donors may be halogeno sugars, sugar
1,2-orthoesters, 1-O-acyl sugars or thioglycosides, and promoters may be
chosen from metal salts or other electrophilic reagents such as silver
oxide, silver carbonate, silver triflate, HgBr.sub.2, Hg(CN).sub.2,
methyl-sulfenyl triflate, and dimethylthiomethylsulfonium triflate. Groups
in the sugar derivatives that are sensitive to the reaction conditions may
be protected. Thus, the hydroxy groups may be protected with acyl groups
such as acetyl or benzoyl, with benzyl, or with a benzylidene group. The
products formed may be purified by methods well known in the art such as
extraction, crystallization or chromatography. The protecting groups may,
if desired, subsequently be (selectively) removed by methods well known in
the art, optionally followed by transformations of the deprotected
positions and further purification.
The lactam nitrogen atom(s) may be introduced via the corresponding
azide(s), which are present in the starting monosaccharide building
blocks. Reduction of the azide to the amine may be carried out by
treatment with sodium borohydride, optionally with addition of nickel
chloride and boric acid, or by treatment with various thiols and hydrogen
sulfide. The reduction may be carried out either before or after the
glycosylation step that introduces a sialic acid residue. However, it is
presently considered preferable to carry out the reduction late in the
synthetic sequence, thereby avoiding protection of the amine formed. The
reaction temperatures may range from -78.degree. C. to +150.degree. C.,
normally from -20.degree. C. to +50.degree. C., such as 0.degree. C. The
reaction time may be from 0.1 to 200 hours, normally 0.1-24 hours such as
20 minutes.
Ring closure of the lactam ring(s) may be carried out by treatment of an
amino-sialic acid methyl ester-saccharide with a suitable solvent such as
pyridine. With the presently known systems, additional catalysts (such as
acids or bases) do no seem to be necessary; however, this might be needed
in other cases. The reaction temperatures range from -78.degree. C. to
+150.degree. C., normally from 0.degree. C. to 50.degree. C. such as room
temperature. The reaction time may be from 0.1 to 200 hours, normally
0.1-24 hours such as 12 hours.
Conversion of the oligosaccharidic lactams into e.g. carrier glycosides may
be carried out using the general methods described inter alia in the paper
by G. Magnusson el al. cited above.
In another aspect, the present invention relates to an antibody which is
directed against a compound of formula I described above. The antibody is
preferably also capable of reacting with the corresponding ganglioside
lactone.
The antibody is advantageously a monoclonal antibody since these tend to be
of a higher specificity (i.e. a closer geometrical "fit" with the antigen
and consequently a higher binding constant) than polyclonal antibodies,
making them useful for accurate diagnostic determinations.
For purposes not requiring monoclonality, the antibody may be a polyclonal
antibody. This may be prepared by injecting a suitable animal (e.g. a
rabbit, monkey, sheep, mouse, goat, rat, pig, horse, or guinea pig) with a
compound of the invention followed by one or more booster injections at
suitable intervals (e.g. two weeks to a month) up to six months before the
first bleeding. Then, while continuing this established immunization
regimen, the animals are bled about one week after each booster
immunization, and antibody is isolated from the serum in a conventional
manner, e.g. as described in Harboe and Ingild, Scand. J. Immun. 2 (Suppl.
1), 1973, pp. 161-164.
The monoclonal antibody may also be produced by other conventional
techniques (e.g. as described by Kohler and Milstein, Nature 256, 1975, p.
495) e.g. by use of a hybridoma cell line, or by clones or subclones
thereof or by cells carrying genetic information from the hybridoma cell
line coding for said monoclonal antibody. The monoclonal antibody may be
produced by fusing cells producing the monoclonal antibody with cells of a
suitable cell line, and selecting and cloning the resulting hybridoma
cells producing said monoclonal antibody. Alternatively, the monoclonal
antibody may be produced by immortalizing an unfused cell line producing
said monoclonal antibody, subsequently growing the cells in a suitable
medium to produce said antibody, and harvesting the monoclonal antibody
from the growth medium. The cells producing the antibodies of the
invention may be spleen cells or lymph cells, e.g. peripheric lymphocytes,
from an immunized animal.
When hybridoma cells are used in the production of antibodies of the
invention, these may be grown in vitro or in a body cavity of an animal.
The antibody-producing cell is injected into an animal such as a mouse
resulting in the formation of an ascites tumour which releases high
concentrations of the antibody in the ascites of the animal. Although the
animals will also produce normal antibodies, these will only amount to a
minor percentage of the monoclonal antibodies which may be purified from
ascites by standard purification procedures such as centrifugation,
filtration, precipitation, chromatography or a combination thereof.
An example of a suitable manner in which the monoclonal antibody may be
produced is as a result of fusing spleen cells from immunized mice (such
as Balb/c mice) with myeloma cells using conventional techniques (e.g. as
described by R. Dalchau, J. Kirkley, J. W. Fabre, "Monoclonal antibody to
a human leukocyte-specific membrane glycoprotein probably homologous to
the leukocyte-common (L-C) antigen of the rat", Eur. J. Immunol. 10, 1980,
pp. 737-744). The fusions obtained are screened by conventional techniques
such as binding assays employing the compounds of the invention.
For some purposes, it may be an advantage that the antibody is a hybrid
antibody which contains a combining site directed against an epitope of
the compound of the invention and further containing another combining
site directed against another epitope of the same antigen, an epitope of
another antigen or an epitope of a pharmaceutical. The term "combining
site" is understood to mean the antigen recognition structure in the
variable region of the antibody molecule. Hybrid antibodies make special
procedures possible for detecting the antigen in a sample and for
targeting a pharmaceutical or other biologically active molecule or
another antigen to the site of the tumour where the reagent has the
greatest effect. In an advantageous embodiment, the other antigen with
which the hybrid antibody is reactive is a differentiation antigen of
cytotoxic T-cells (cf. Staerz et al., Nature 314, 1985, p. 628). The
pharmaceutical with which the hybrid antibody may be reactive is
preferably selected from cytotoxic or antineoplastic agents (cf. Collier,
R. J. and Kaplan, D. A., Scientific American 251, 1984, p. 44), see the
discussion below.
The hybrid antibody may be produced by hybrids between two monoclonal cell
lines producing the two relevant antibodies or may be produced by
chemically linking fragments of the two antibodies.
The invention further relates to an antibody which, for various purposes,
vide below, is an anti-idiotypic antibody, i.e. an antibody directed
against the site of an antibody which is reactive with an epitope on the
antigen, i.e. the compound of the invention. The anti-idiotypic antibody
is directed against an antibody which is reactive with the compound of the
invention. The anti-idiotypic antibody may be prepared by a similar method
to that outlined above for the monoclonal or polyclonal antibody. The
invention also relates to an anti-anti-idiotypic antibody directed against
the anti-idiotypic antibody defined above.
An antibody directed against a compound of the invention as well as an
anti-anti-idiotypic antibody defined above may in principle be used in the
purification of compounds of formula I or of the anti-idiotypic antibodies
described above by affinity chromatography.
In a further important aspect, the present invention relates to a
diagnostic agent which comprises a compound of formula I as described
above, an antibody of the invention as described above, an anti-idiotypic
antibody as described above, or an anti-anti-idiotypic antibody as
described above.
An antibody directed against the compound of the invention as well as an
anti-anti-idiotypic antibody defined above may be used for in vitro
diagnosis of cancer. Thus, for instance a biopsy sample may be treated
with the antibody or anti-anti-idiotypic antibody followed by detection of
bound antibody as described below. The anti-idiotypic antibody may, like
the compound of formula I of the invention itself, be used to detect the
presence in body fluids of an antibody against the compounds of the
invention and thus to assess the strength of an immune response or whether
further antibody treatment is necessary.
It is preferred for most purposes to provide the antibody with a label in
order to detect bound antibody. In a double antibody ("sandwich") assay,
at least one of the antibodies may be provided with a label in a
well-known manner. Substances useful as labels in the present context may
be selected from enzymes, fluorescent substances, radioactive isotopes and
ligands such as biotin.
It should be noted that practically all methods or applications based on
intact antibodies could instead be performed using fragments of the
antibodies, e.g. F(ab').sub.2 or Fab fragments (cf. Delaloye, B. et al.,
J. Clin. Invest. 87, 1986, p. 301).
The invention further relates to a vaccine which comprises a compound of
formula I according to the invention and a physiologically acceptable
excipient or adjuvant. In connection with use in such vaccines, in it is
preferred that the compound of the invention is one in which R.sup.10 is a
carrier CA or a group (Sugar).sub.n as defined above and in which the
reducing-end terminal sugar unit is glycosidically bound to a carrier CA.
In particular, it is preferred that the carrier CA comprises a protein
carrier or is a lipid group as described above.
Generally speaking, the vaccine should be made so as to allow an optimal
stimulation of the relevant parts of the immune system, i.e to present the
immunogenic agent for a period of time and in a form being optimal with
respect to the recognition, the uptake or any other interaction or
processing necessary for the stimulation.
If the carrier CA is a lipid group, a particularly interesting embodiment
of the vaccine is that in which the compound of formula I forms part of a
supramolecular aggregate. The term "supramolecular aggregate" is intended
to mean suspensions, colloids, emulsions, or solutions (depending on size)
of particles on whose surface the compound of the invention is located in
a manner where it is able to interact with the surrounding environment so
as to enable the immune system to detect its presence and to elicit an
immune response. It is contemplated that the particles may be any of a
number of especially biodegradable structures such as biodegradable
polysaccharide particles, e.g. dextran particles, "liquid" particles
comprising membranes consisting of surfactant layers having the compound
of formula I embedded in the fluid membrane. Examples of such "liquid"
particles are liposomes, micelles, or cubic phase particles.
In yet another aspect, the invention relates to a pharmaceutical
composition for the treatment of human carcinoma having ganglioside
lactones as carcinoma-associated antigens, which comprises an antibody
according to any of claims 10-16 and a pharmaceutically acceptable
excipient.
The excipient employed in the composition of the invention may be any
pharmaceutically acceptable vehicle. This vehicle may be any vehicle
usually employed in the preparation of injectable compositions, e.g. a
diluent, suspending agent etc. such as isotonic or buffered saline. The
composition may be prepared by mixing a therapeutically effective amount
of the antibody with the vehicle in an amount resulting in the desired
concentration of the antibody in the composition.
In some cases it may be advantageous to couple the antibody to a carrier,
in particular a macromolecular carrier. Such macromolecular carriers may
be any of those described above in connection with the compounds of the
invention. Thus, the macromolecular carrier is usually a polymer to which
the toxin is bound by hydrophobic non-covalent interaction, such as a
plastic, e.g. polystyrene, or a polymer to which the antigen or antibody
is covalently bound, such as a polysaccharide, or a polypeptide, e.g.
bovine serum albumin, ovalbumin or keyhole limpet hemocyanin. Furthermore,
the macromolecular carrier may advantageously be selected from a
pharmaceutical, e.g. a cytotoxic or antineoplastic agent, to which the
antibody is coupled. The macromolecular carrier may also be another
antibody directed against a cytotoxic effector mechanism, e.g. cytotoxic
cells. The macromolecular carrier should preferably be nontoxic and
non-allergenic. The antibody may be multivalently coupled to the
macromolecular carrier as this may provide an increased immunogenicity of
the composition.
For oral administration, the composition may be in the form of a tablet,
capsule, granulate, paste, gel, mixture or suspension optionally provided
with a sustained-release coating or a coating which protects the antigen
(i.e. the compound of the invention) from passage through the stomach.
Solid formulations, i.e. granulates, tablets and capsules, may contain
fillers, e.g. sugars, sorbitol, mannitol and silicic acid; binders, e.g.
cellulose derivatives such as carboxymethyl cellulose and
polyvinylpyrrolidone; disintegrants, e.g. starch, sodium bicarbonate and
calcium carbonate; lubricants, e.g. magnesium stearate, talc and calcium
stearate. Semisolid formulations, i.e. pastes or gels, may comprise a
gelling agent such as an alginate, gelatin, carrageenan, tragacanth gum
and pectin, a mineral oil such as liquid paraffin, a vegetable oil such as
corn oil, sunflower oil, rape oil and grape kernel oil, as well as a
thickener such as a starch, gum, gelatin, etc. Liquid formulations, i.e.
mixtures and suspensions, may comprise an aqueous or oily vehicle, e.g.
water, or a mineral oil such as liquid paraffin, a vegetable oil such as
corn oil, sunflower oil, rape oil, grape kernel oil, etc. The antigen of
the invention may be suspended in the liquid vehicle in accordance with
usual practice.
The sustained-release coating may, e.g., be an enteric coating which may be
selected from shellac, cellulose acetate esters such as cellulose acetate
phthalate, hydroxypropylmethyl cellulose esters such as
hydroxypropylmethyl cellulose phthalate, polyvinyl acetate esters such as
polyvinyl acetate phthalate, and polymers of methacrylic acid and
(meth)acrylic acid esters.
In particular injectable compositions may be formulated using the types of
particle suspensions, colloids, emulsions, or solutions described above in
connection with the vaccine of the invention.
The composition may also be adapted for rectal administration, e.g. as a
suppository. Such a suppository may contain conventional excipients such
as cocoa butter or other glycerides.
Furthermore, the invention relates to the use of a compound according to
the invention or an anti-idiotypic antibody of the invention for preparing
a medicament for the treatment of human cancer, or to the use of an
antibody or anti-antiidiotypic antibody according to the invention and for
preparing a medicament for the treatment of human carcinoma.
Therapy of cancers, in particular carcinomas, the cells, in particular the
cell membranes, of which comprise ganglioside lactones in concentrations
higher than in normal cells may be carried out by a variety of procedures
known to those skilled in the art. An antibody against the compounds of
the invention (in particular a human monoclonal antibody for the reasons
stated above) may be injected into cancer patients to combat the tumour
directly or via various effector mechanisms, e.g. complement-mediated
cytotoxicity or antibody-dependent cell-mediated cytotoxicity.
In another embodiment, the antibody may be utilized in a drug targeting
approach. Thus, the antibody may be modified prior to injection into the
patient as indicated above, e.g. by coupling to pharmaceuticals (thus
transporting those to the site of their activity), or to another antibody
directed against a cytotoxic effector mechanism such as antigens on
cytotoxic T-cells or on other cytotoxic cells. It is contemplated that a
hybrid antibody containing one combining site for the antigen compound of
the invention and another combining site for another antigen or for a
pharmaceutical as described above may also advantageously be used to
provide a two-way attack on the tumour in question.
In principle, the targeting of the pharmaceutical may be carried out using
either a hybrid antibody to which the pharmaceutical is already coupled by
antigen-antibody-reaction or using a hybrid antibody which does not have
any pharmaceutical coupled to it yet. In the first case, the antibody will
upon administration seek out the ganglioside lactones on the cancer cell
surfaces bringing the pharmaceutical with it whereupon the pharmaceutical
will be able to exert its effect. In the second case, it will be necessary
for the pharmaceutical to be administered separately, either before or
after the antibody, and the antibody will then react with the
pharmaceutical in the body on the surface of a cancer cell or in the blood
stream.
The pharmaceutical which is coupled to the antibody may typically be an
anti-cancer agent such an alkylating agent, e.g. melphalan, chlorambucil,
busulfan, cisplatin, thiotepa, an antimetabolite such as methotrexate,
fluracil, azathioprin, an antimitoticum, typically vincristine,
vinblastine, or an antibiotic such as doxorubicin, daunorubicin or
bleomycin. The medicament may also comprise bacterial or other toxins.
The compounds of the invention may be used for immunization in order to
provoke an anti-cancer immune response in the body. For this reason, the
invention further relates to a vaccine which comprises a compound of
formula I according to the invention and a physiologically acceptable
excipient. The compounds may further be used in vitro for raising effector
cells against cancer by culturing the compound with e.g. leukocytes from a
cancer patient.
It is further contemplated that the compounds or the pharmaceutical
compositions of the invention may be used in a method of inhibiting
cell-cell or cell-virus interaction, in particular in connection with
bacterial or viral attachment to cells, inflammation, or metastasis of
tumours, in human beings or animals comprising administering a compound or
a pharmaceutical composition according to the invention. The basis for the
method is the fact that one of the functions of gangliosides on cell
surfaces is to function as receptors, and it is known that in interactions
between on the one hand e.g. bacteria or viruses and on the other hand
mammalian cells, gangliosides function as recognition receptors in that
they interact with epitopes on the surfaces of the bacteria or viruses and
help these entities to adhere to the cell surface or even to actually
invade the cell. Thus, the administration of compounds of the invention
may serve to "saturate" the epitopes on the bacteria, viruses or
metastasis cells and may therefore result in a reduced ability of the
epitopes to interact with the natural receptors.
The compounds of the invention may further be used in extra-corporal
devices for removing circulating anti-tumour antibody or immune complexes
or for purifying antibody preparations. Consequently, the invention also
relates to a method of purifying antibodies according to the invention
comprising contacting a solution containing the antibodies with a compound
of the formula I with which the antibody is able to react, the compound
being coupled to or comprising a solid support (such as the stationary
phase, e.g. silica gel or dextran, in a affinity chromatographical
process); removing the solution from the support; and releasing the
antibody from the support. The release of the bound antibody from the
support may be carried out by methods well known in the art, such as
described by Maniattis et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbour, 1982, e.g. change in pH, salinity, or ionic strength,
displacement with solutions of antigen-like compounds followed by
dialysis, etc.
As a natural continuation of the possibility of using the compounds of the
invention in a vaccine, the invention also relates to a method of treating
or of boosting the immune protection against human cancer having
ganglioside lactones as cancer-associated antigens, comprising
administering a compound according to the invention in an amount capable
of inducing the formation of antibodies against the compound.
The antibodies against the compounds of the invention may furthermore be
administered to provoke an anti-idiotypic or anti-anti-idiotypic immune
response. An anti-idiotypic antibody (whether monoclonal or polyclonal or
a fragment of these) raised against an antibody reacting with the
compounds of the invention may express epitopes similar to those of the
antigen compound and may therefore be used in a similar fashion for
immunization to elicit an anti-carcinoma immune response. Such antibodies
may further be used in extra-corporal devices as described above for the
primary antibody.
Anti-anti-idiotypic antibodies may be used in ways similar to those
described for primary anti-tumour antibodies.
A further embodiment of a diagnostic method of the invention relates to a
method of localizing tumours (in particular carcinomas) in vivo by means
of the antibody of the invention. This method comprises administering a
diagnostically effective amount of an antibody of the invention which is
labelled so as to permit detection thereof, and determining the sites of
localization of bound antibody. The antibody may be labelled by means of a
radioactive isotope, in particular a physiologically tolerable isotope
such as technetium, and subsequently injected and localized by known
methods, e.g. a gamma ray detector of a suitable configuration (cf. Mach,
J.-P. et al., Nature 248, 1974, p. 704).
BRIEF DESCRIPTION OF DRAWINGS
The invention is illustrated by the drawing, on which
FIG. 1 shows a two-dimensional rendering of the energy-minimized
three-dimensional structure of GM.sub.3 -lactone methyl glycoside;
FIG. 2 shows a two-dimensional rendering of the energy-minimized
three-dimensional structure of the methyl glycoside of the GM.sub.3
-lactam prepared in example 1; and
FIG. 3 shows a superimposition of the two structures shown in FIGS. 1 and
2.
FIGS. 4 a-d shows inhibition curves for the binding of some monoclonal
antibodies raised against GM.sub.3 -lactam-BSA conjugate, cf. Example 5
below.
EXAMPLES
The invention is further illustrated by the following examples. The various
starting materials, intermediates and products 1-29 used or prepared in
examples 1-4 are illustrated below.
##STR10##
Example 1
Synthesis of the neoglycoprotein GM.sub.3 -lactam-BSA (18) was performed
using the starting materials 1,2, and 10. The intermediates 3-9 and 11-17
were isolated and characterized.
A) 2-(Trimethylsilyl)ethyl
2,3,6-tri-O-benzyl-4-O-(3,4,6-tri-O-acetyl-2-azido-2-deoxy-.alpha./.beta.-
D-galactopyranosyl)-.beta.-D-glucopyranoside (3.alpha..beta.)
Compound 2 (Jansson et al. J. Org. Chem. 1988, 53, 5629; 4.5 g, 8.22 mmol),
1 (2.9 g, 7.36 mmol), and dry molecular sieves (4 .ANG., 3 g) were
dissolved in dry dichloromethane (50 mL) and the mixture was stirred under
nitrogen for 60 min. Silver silicate (van Boeckel, C. A. A., Beetz, T.
Rec. Trav. Chim. Pays-Bas, 1987, 106, 596; 8 g) was added and the mixture
was stirred vigorously at room temperature for 18 h, then filtered through
Celite and concentrated. The residue was chromatographed (SiO.sub.2 ;
heptane/EtOAc gradient, 6:1.fwdarw.4:1) to give 3.alpha..beta. (3.87 g,
61%; .alpha./.beta. .apprxeq.8:92).
.sup.1 H-NMR data (CDCl.sub.3) .delta. 5.19 (d, 1H, J=3.37 Hz, H-4'.beta.),
4.60 (dd, 1H, J=3.27, 10.8 Hz, H-3'.beta.), 4.40 (d, 1H, J=7.6 Hz,
H-l.beta.), 4.39 (d, 1H, J=8.1 Hz, H-l'.beta.), 2.09, 2.04, 1.99 (3s, 3H
each, OAc.beta.), 1.05 (m, 2H, CH.sub.2 Si), 0.04 (s, 9H, SiMe.sub.3).
.sup.13 C-NMR (CDCl.sub.3) .delta. 103.3, 103.0, 100.7, 97.6.
B) 2-(Trimethylsilyl)ethyl
2,3,6-tri-O-benzyl-4-O-(2-azido-2-deoxy-.alpha./.beta.-D-galactopyranosyl)
-.beta.-D-glucopyranoside (4.alpha..beta.)
Compound 3.alpha..beta. (3.7 g, 4.28 mmol) was treated with methanolic
sodium methoxide (0.2M, 50 mL) at room temperature for 6 h. The mixture
was neutralized with Duolite (H.sup.+) resin, filtered and the solvent was
removed to give crude 4.alpha..beta. (3.06 g, 97%), which was used in the
next step without purification.
C) 2-(Trimethylsilyl)ethyl 2,3,6-tri-O-benzyl-4-O-(2-azido-2-deoxy-3,4- and
-4,6-O-isopropylidene-.alpha./.beta.-D-galactopyranosyl)-.beta.-D-glucopyr
anoside (5.alpha..beta. and 6.beta.)
Compound 4.alpha..beta. (750 mg, 1.01 mmol) and (.+-.)-camphorsulfonic acid
(15 mg) were dissolved in 2,2-dimethoxypropane (25 mL) and the mixture was
stirred at room temperature for 48 h. Triethylamine (2 mL) was added and
the mixture was co-concentrated with toluene (4.times.20 mL) to remove
traces of amine. The residue was dissolved in methanol/water (10:1, 44 mL)
and the mixture was refluxed (bath temperature: 85.degree. C.) for 3
hours, then co-concentrated with toluene (3.times.30 mL). The residue was
chromatographed (SiO.sub.2 ; heptane/EtOAc gradient, 5:1.fwdarw.1:2) to
give (in order of elution):
5.alpha. (61 mg, 8%; R.sub.f =0.25, SiO.sub.2, heptane/EtOAc 2:1);
[.alpha.]D.sup.22 +71.degree. (c 1.7, CDCl.sub.3);
.sup.1 H-NMR data (CDCl.sub.3) .delta. 5.62 (d, 1H, J=3.7 Hz, H-l'), 4.43
(d, 1H, J=7.8 Hz, H-l), 1.42, 1.29 (2s, 3H each, Me.sub.2 C), 1.05 (m, 2H,
CH.sub.2 Si), 0.05 (s, 9H, SiMe.sub.3), and
5.beta./6.beta. (19:2; 620 mg, 78%; Rf=0.13, SiO.sub.2 heptane/EtOAC, 2:1);
[.alpha.].sub.D.sup.22 +38.degree. (c 1.6, CDCl.sub.3);
.sup.1 H-NMR data for 5.beta. (CDCl.sub.3) .delta. 4.40 (d, 1H, J=7.8 Hz,
H-l), 4.21 (d, 1H, J=8.5 Hz, H-l'), 1.55, 1.33 (2s, 3H each, Me.sub.2 C),
1.04 (m, 2H, CH.sub.2 Si), 0.04 (s, 9H, SiMe.sub.3).
D) 2-(Trimethylsilyl)ethyl
2,3,6-tri-O-benzyl-4-O-(2-azido-6-O-benzyl-2-deoxy-3,4-O-isopropylidene-.b
eta.-D-galactopyranosyl)-.beta.-D-glucopyranoside (7.beta.) and
2-(Trimethylsilyl) ethyl
2,3,6-tri-O-benzyl-4-O-(2-azido-4-O-benzyl-2-deoxy-4,6-O-isopropylidene-.b
eta.-D-galactopyranosyl)-.beta.-D-glucopyranoside (8.beta.)
The mixture 5.beta.6.beta. (2.35 g, 3.02 mmol) was dissolved in
dimethylformamide (40 mL) and sodium hydride (50% oil coating; 0.3 g, 6.2
mmol) was added with stirring at room temperature. After 1 h, benzyl
bromide (0.6 mL, 5 mmol), was added and the mixture was stirred at room
temperature over night. Methanol (5 mL) was added dropwise,
dichloromethane (300 mL) was added, the mixture was washed with water
(5.times.200 mL), dried (Na.sub.2 SO.sub.4) and concentrated. The residue
was chromatographed (SiO.sub.2 ; heptane/EtOAc gradient, 20:1.fwdarw.1:1)
to give (in order of elution):
7.beta. (2.24 g, 86%; Rf 0.31, SiO.sub.2, heptane/EtOAc 3:1)
[.alpha.].sub.D.sup.25 +21.degree. (c 1.4, CDCl.sub.3);
.sup.1 H-NMR data (CDCl.sub.3) .delta. 4.40 (d, 1H, J=7.8 Hz, H-l), 4.29
(d, 1H, J=8.3 Hz, H-l'), 1.54, 1.36 (2s, 3H each, Me.sub.2 C), 1.04 (m,
CH.sub.2 Si), 0.04 (s, 9H, SiMe.sub.3), and
8.beta. (221 mg, 8%; Rf 0.15, SiO.sub.2, heptane/EtOAc 3:1);
.sup.1 H-NMR data (CDCl.sub.3) .delta. 4.40 (d, 1H, J=7.9 Hz, H-1), 4.26
(d, 1H, J=8.1 Hz, H-1'), 3.10 (dd, 1H, J=3.6, 10.2 Hz, H-3'), 1.45, 1.37
(2s, 3H each, Me.sub.2 C), 1.04 (m, CH.sub.2 Si).
E) 2-(Trimethylsilyl)ethyl
2,3,6-tri-O-benzyl-4-O-(2-azido-6-O-benzyl-2-deoxy-.beta.-D-galactopyranos
yl)-.beta.-D-glucopyranoside (9.beta.)
Compound 7.beta. (2.20 g, 2.53 mmol), was dissolved in acetic acid/water
(50 mL; 85:15) and the mixture was stirred at 85.degree. C. for 90 min.,
then co-concentrated with toluene (5.times.20 mL). The residue was
chromatographed (SiO.sub.2 ; heptane/EtOAc gradient, 4:1.fwdarw.2:1) to
give 9.beta. (1.96 g, 94%); (.alpha.).sub.D.sup.22 +19.degree. (c 1.1,
CDCl.sub.3);
.sup.1 H-NMR data (CDCl.sub.3) .delta. 4.40 (d, 1H, J=7.6 Hz, H-l), 4.31
(d, 1H, J=8.1 Hz, H-l'), 2.76 (d, 1H, J=3.7 Hz, OH), 2.56 (d, 1H, J=8.0
Hz, OH), 1.04 (m, 2H, CH.sub.2 Si), 0.04 (s, 9H, SiMe.sub.3).
F) 2-(Trimethylsilyl)ethyl O-(methyl
5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-di-deoxy-D-glycero-.alpha.-D-galact
o-2-nonulpyranosylonate)-(2.fwdarw.3)-O-(2-azido-2-deoxy-6-O-benzyl-.beta.-
D-galactopyranosyl)-(1.fwdarw.4)-2,3,6-tri-O-benzyl-.beta.-D-glucopyranosid
e (11)
Compound 9.beta. (1.98 g, 2.39 mmol) and
O-ethyl-S-[methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-di-deoxy-.alpha.-
D-glycero-D-galacto-2-nonulopyranosyl)onate] (Marra, A. Sinay, P.
Carbohydr. Res. 1989, 187, 35; 10; 1.71 g, 2.87 mmol) were dissolved in
freshly distilled dry acetonitrile/dichloromethane (70 mL, 3:2) and
molecular sieves (3 .ANG.; 4 g, activated by heating) was added. The
mixture was stirred under nitrogen at room temperature for 90 min. Silver
triflate (0.738 g; 2.87 mmol) was added and the mixture was cooled to
-78.degree. C. and stirred for 20 min. Methylsulfenyl bromide (Dasgupta,
F., Garegg, P. J. Carbohydr. Res. 1988, 177, c13; 0.365 g, 2.87 mmol) in
1,2-dichloroethane (0.77 mL) was added (syringe) to the reaction mixture
and stirring was continued at -78.degree. C. for 2 h. Di-isopropylamine (1
mL) was added and the mixture was stirred at -78.degree. C. for 1 h, then
filtered through Celite with dichloromethane (300 mL, .about.20.degree.
C.). The mixture was washed with saturated aqueous sodium hydrogen
carbonate and water, dried (Na.sub.2 SO.sub.4) and concentrated. The
residue was chromatographed (SiO.sub.2 ; toluene/EtOH gradient,
45:1.fwdarw.25:1) to give impure 11. A second chromatography (SiO.sub.2)
using different solvents gave the following compounds:
i) with hexane/EtOAc (1:1), 9.beta. (0.81 g, 41%);
ii) with toluene/EtOAc (5:4.fwdarw.1:1.fwdarw.1:2), 2"-3'-.beta.-linked
trisaccharide (0.15 g, 5%);
iii) with toluene/EtOAc (1:3), compound 11 (1.63 g, 52%);
iv) with EtOAc, the 2,3-elimination product of 10 (0.59 g, 43%).
Compound 11 had: [.alpha.].sub.D.sup.22 -13.degree. (c 1.1, CDCl.sub.3);
.sup.1 H-NMR data (CDCl.sub.3) .delta. 5.54 (m, 1H, H-8"), 5.32 (bd, 1H,
H-7-), 5.19 (bd, 1H, NH), 4.50 (d, 1H, J=8.2 Hz, H-l'), 4.40 (d, 1H, J=7.3
Hz, H-l), 4.17 (dd, 1H, J=3.2, 10.0 Hz, H-3'), 3.75 (s, 3H, COOMe), 2.67
(dd, 1H, J=4.4, 12.9 Hz, H-3"eq), 2.10, 2.06, 2.05, 1.98, 1.89, (5s, 3H
each, OAc, NHAc), 1.04 (m, 2H CH.sub.2 Si), 0.03 (s, 9H, SiMe.sub.3);
.sup.13 C-NMR data (CDCl.sub.3) .delta. 170.9, 170.6, 170.2, 170.1, 170.0,
168.4, 139.3, 138.7, 138.5, 138.1, 128.3, 128.2, 128.1, 127.6, 127.5,
127.46, 127.4, 127.1, 103.1, 100.6, 97.5, 83.1, 82.1, 76.9, 75.2, 74.9,
74.8, 73.3, 73.2, 72.5, 72.2, 69.0, 68.7, 68.4, 68.3, 674, 67.1, 66.9,
62.7, 62.4, 53.1, 49.3, 37.0, 23.2, 21.2, 20.9, 20.7, 20.6, 18.5, -1.4.
G) TMSEt GM.sub.3 -lactam (12)
Compound 11 (400 mg,0.306 mmol), nickel chloride (NiCl.sub.2 /6 H.sub.2 O;
1.37 g, 5.75 mmol), and boric acid (648 mg, 11.1 mmol) were dissolved in
ethanol (30 mL) and the mixture was stirred and cooled to 0.degree. C. A
solution of sodium borohydride (308 mg, 8.14 mmol) in ethanol (20 mL) was
added dropwise during 10 min. After 10 min., the mixture was concentrated
and the residue was dissolved in dichloromethane (50 mL). The mixture was
washed with saturated aqueous sodium hydrogen carbonate and water, dried
(Na.sub.2 SO.sub.4) and concentrated. The residue was treated with
methanolic sodium methoxide (0.05M, 5 mL) at room temperature over night,
then neutralized with acetic acid and co-concentrated with toluene. The
residue was stirred with pyridine (5 mL) at room temperature over night
and co-concentrated with toluene. The residue was filtered through silica
(CHCl.sub.3 /MeOH 10:1) and the filtrate was concentrated. The residue was
hydrogenolyzed (H.sub.2, Pd/C., 10%, 1 atm.) in acetic acid (10 mL) at
room temperature over night. The mixture was filtered through Celite and
the filtrate was concentrated. The residue was chromatographed (SiO.sub.2
; CHCl.sub.3 /MeOH/H.sub.2 O gradient, 10:4:1.fwdarw.-10:6:1) to give 12
(118 mg, 54%); [.alpha.].sub.D.sup.24 -22.degree. (c 0.7, MeOH);
.sup.1 H-NMR data (D.sub.2 O) .delta. 4.69 (d, 1H, J=8.0 Hz, H-l'), 4.47
(d, 1H, J=8.1 Hz, H-l), 4.32 (m, 1H, H-4"), 3.23 (t, 1H, J=9.0 Hz, H-2),
2.59 (dd, 1H, J=5.3, 13.4 Hz, H-3"eq), 2.02 (s, 3H, NHAc), 1.67 (dd, 1H,
J=11.4, 12.9 Hz, H-3"ax), 1.00 (m, 2H, CH.sub.2 Si), 0.00 (s, 9H,
SiMe.sub.3);
13C-NMR (D.sub.2 O) .delta. 175.9, 169.6, 102.3, 100.7, 98.8, 78.9, 78.8,
77.0, 74.9, 74.8, 73.9, 73.2, 71.0, 69.3, 68.6, 67.5, 66.3, 64.1, 61.8,
61.5, 52.6, 51.6, 40.1, 22.9, 18.4, -1.7.
H) TMSEt GM.sub.3 -lactam-Ac (13)
Compound 12 (113 mg, 0.158 mmol) was treated with acetic anhydride/pyridine
(1:1,5 mL) at room temperature for 24 h, then co-concentrated with
toluene. The residue was chromatographed (SiO.sub.2 ; toluene/ethanol,
5:1) to give 13 (170 mg, 98%); [.alpha.].sub.D.sup.24 -32.degree. (c 0.8,
CDCl.sub.3);
.sup.1 H-NMR data (CDCl.sub.3) .delta. 5.61 (bd, 1H, J=10.5 Hz, H-7"), 5.46
(bdt, 1H, H-4"), 4.45 (dd, 1H, J=7.9, 9.2 Hz, H-2), 4.54 (d, 1H, J=7.8 Hz,
H-l), 4.40 (d, 1H, J=8.6 Hz, H-l'), 2.40 (dd, 1H, J=5.6 and 13.4 Hz,
H-3"eq), 2.20, 2.18, 2.09, 2.09, 2.08, 2.06, 2.04, 2.03, 2.00, 1.89 (9s,
3H each, OAc, NHAc), 1.79 (dd, 1H, J=11.4, 12.9 Hz, H-3"ax), 0.90 (m, 2H,
CH.sub.2 Si), 0.00 (s, 9H, SiMe.sub.3).
I) Chloro GM.sub.3 -lactam-Ac (14)
Compound 13 (167 mg, 0.153 mmol) and dichloromethylmethyl ether (0.105 mL,
1.18 mmol) were dissolved in dry chloroform (4 mL) and freshly fused zinc
chloride (.apprxeq.20 mg) was added. The mixture was stirred at room
temperature over night, chloroform (25 mL) was added, the mixture was
washed with saturated aqueous sodium hydrogen carbonate and water, dried
(Na.sub.2 SO.sub.4) and concentrated to give crude 14, which was used
without purification.
.sup.1 H-NMR data (CDCl.sub.3) .delta. 6.20 (d, 1H, J=3.9 Hz, H-l).
J) 2-Bromoethyl GM.sub.3 -lactam-Ac (15)
Compound 14 (156 mg, 0.154 mmol) was added dropwise to a stirred mixture of
2-bromoethanol (0.1 mL; 1.4 mmol), silver trifluoromethane sulfonate (52
mg, 0.2 mmol), and molecular sieve (3 .ANG., 0.1 g) in dichloromethane (2
mL) at -28.degree. C. under nitrogen. After 4 h, the cooling bath was
removed and the mixture was left over night, then filtered through Celite,
washed with saturated aqueous sodium hydrogen carbonate and water, dried
(Na.sub.2 SO.sub.4), and concentrated. The residue was chromatographed
(SiO.sub.2 ; toluene/ethanol, 10:1) to give 15 (90 mg, 54%) as an
.alpha.,.beta.-mixture (15:85);
[.alpha.].sub.D.sup.25 -27.degree. (c 1.2, CDCl.sub.3);
.sup.1 H-NMR data (CDCl.sub.3) .delta. 4.90 (dd, 1H, J=8.0, 9.4 Hz, H-2),
4.78 (d, 1H, J=8.1 Hz, H-l), 3.42 (bt, 2H, OCH.sub.2 CH.sub.2), 2.40 (dd,
1H, J=5.6, 13.2 Hz, H-3"eq), 2.20, 2.15, 2.13, 2.07, 2.06, 2.05, 2.00,
1.89, (8s, 30H, OAc, NHAc).
K) Spacer-GM.sub.3 -lactam-Ac (16)
Compound 15 (90 mg, 0.082 mmol), cesium carbonate (32 mg, 0.10 mmol), and
dimethylformamide (4.5 mL) were stirred at room temperature under nitrogen
for 10 min. Methyl 3-mercaptopropionate (37 .mu.L, 0.33 mmol) was added,
the mixture was stirred for 2.5 h, dichloromethane (30 mL) was added, and
the mixture was washed with water, dried (Na.sub.2 SO.sub.4) and
concentrated. The residue was chromatographed (SiO.sub.2 ; toluene/ethanol
gradient, 15:1.fwdarw.10:1) to give 16 (77 mg, 82%) as an
.alpha.,.beta.-mixture (.about.15:85); [.alpha.].sub.D.sup.22 -23.degree.
(c 1.1, CDCl.sub.3);
.sup.1 H-NMR data (CDCl.sub.3) .delta. 4.88 (dd, 1H, J=8.0, 9.5 Hz, H-2),
4.63 (d, 1H, J=7.9 Hz, H-l), 3.69 (s, 3H, COOMe), 2.78, 2.68, 2.60 (t, 2H
each, CH.sub.2), 2.38 (bdd, 1H, H-3"eq), 2.19, 2.15, 2.11, 2.07, 2.068,
2.06, 2.04, 2.00, 1.99, 1.89, (10s, 3H each, OAc, NHAc);
.sup.13 C-NMR (CDCl.sub.3) .delta. 172.171.4, 171.2, 170.8, 170.4, 170.3,
170.1, 169.9, 169.8, 169.7, 167.7, 100.5, 100.2, 97.8, 77.2, 76.7, 75.2,
75.1, 73.0, 72.3, 72.1, 71.7, 71.4, 70.4, 69.7, 69.4, 67.3, 65.1, 62.6,
61.0, 51.8, 50.9, 48.7, 37.3, 34.7, 31.5, 29.7, 27.4, 23.1, 21.1, 21.0,
21.0, 20.8, 20.7, 20.67, 20.6, 20.5, 14.6.
L) Spacer-GM.sub.3 -lactam (17 )
Compound 16 (49 mg, 0.043 mmol) was dissolved in methanolic sodium
methoxide (0.02M, 2 mL) and the mixture was stirred at room temperature
for 4 h, then neutralized with Duolite H.sup.+ resin, filtered and
concentrated. The residue was chromatographed (SiO.sub.2 ; CHCl.sub.3
/MeOH/H.sub.2 O, 10:5:1) to give 17 (28 mg, 86%) as an
.alpha.,.beta.-mixture (.about.1:8); [.alpha.].sub.D.sup.24 0.1.degree. (c
0.5, MeOH);
.sup.1 H-NMR data (CDCl.sub.3) .delta. 4.92 (d, 1H, J=4.1 Hz, H-l.alpha.),
4.69 (d, 1H, J=8.1 Hz, H-l'), 4.48 (d, 1H, J=8.1 Hz, H-l.beta.), 4.32 (m,
1H, H-4"), 3.70 (s, 3H, COOMe), 3.30 (t, 1H, J=8.1 Hz, H-2), 2.85, 2.81
(bt, 2H each, CH.sub.2), 2.71 (bt, 2H, CH.sub.2), 2.57 (dd, 1H, J=5.4,
13.2 Hz, H-3"eq), 2.02 (s, 3H, NHAc) , 1.67 (dd, 1H, J=10.3, 13.2 Hz,
H-3"ax);
.sup.13 C-NMR data (D.sub.2 O) .delta. 175.9, 169.6, 103.1, 100.7, 98.8,
78.74, 78.71, 77.0, 75.0, 74.6, 73.8, 73.2, 71.0, 69.9, 68.6, 66.2, 64.1,
61.8, 61.5, 53.1, 52.6, 51.6, 40.1, 35.0, 31.6, 27.3, 22.9, 13.0, 12.9.
M) GM.sub.3 -lactam-BSA-conjugate (18)
Compound 17 (22 mg, 28.9 .mu.mol), and hydrazine hydrate (85%, 0.25 mL)
were dissolved in ethanol (2 mL) and the mixture was stirred at room
temperature over night and concentrated. The residue was dissolved in
water and freeze-dried. The resulting hydrazide was dissolved in
dimethylsulfoxide (0.5 mL) and hydrogen chloride in dioxane (4M, 53 .mu.L)
was added followed by a solution of tert. butylnitrite (9 .mu.L, 75
.mu.mol) in dimethylsulfoxide (50 .mu.L). The mixture was stirred at room
temperature for 30 min. and a solution of sulfamic acid (5 mg, 55 .mu.mol)
in dimethylsulfoxide (50 .mu.L) was added. After 15 min. the mixture was
added dropwise with stirring to a solution of bovine serum albumin (27 mg,
0.41 .mu.mol) in sodium tetraborate-potassium hydrogen-carbonate buffer (1
mL, 0.08M Na.sub.2 B.sub.4 O.sub.7 and 0.35M KHO.sub.3). The pH was
maintained at 8.5-9.5 by addition of sodium hydroxide solution (1M). The
mixture was stirred at 4.degree.-15.degree. C. for 1 h and at room
temperature over night, then dialyzed against distilled water for 4 d and
freeze dried to give 18 (33 mg). The degree of binding (mol of 18 per mol
of BSA) was 21 according to sulfur combustion analysis.
Example 2
Preparation of TMSEt GM.sub.2 -lactam (22)
A) Compound 19
Compound 11 (1 g, 0.768 mmol) in ethanol (60 ml) was cooled to
.about.0.degree. C. NiCl.sub.2,6H.sub.2 O (3.42 g, 14.4 mmol) and H.sub.3
BO.sub.3 (1.72 g, 27.8 mmol) in ethanol (60 ml) was added dropwise during
15 min. with stirring. Stirring and cooling were continued for another 15
min. The reaction mixture was concentrated to dryness, dissolved in
CH.sub.2 Cl.sub.2 (.about.150 ml) and washed with a saturated solution of
NaHCO.sub.3 and water, respectively. The organic layer was dried (Na.sub.2
SO.sub.4) and evaporated to dryness. The residue was taken up in pyridine
(20 ml) and stirred at 85.degree. C. for 48 hours. Evaporation and
co-evaporation with toluene (10 ml.times.3) followed by chromatography
(toluene-EtOAc 1:2) gave pure 19 (677 mg, 70.8%) as an amorphous powder
having [.alpha.].sub.D.sup.22 -5.degree. (c 1.1, CHCl.sub.3).
.sup.1 H-NMR (CDCl.sub.3) .delta. 5.63 (m, 1H, H-4"), 5.33 (d, 1H, J=10.2
Hz, AcNH), 5.27 (dd, 1H, J=2.20, 5.86 Hz, H-7), 5.16 (m, 1H, H-8"), 4.48
(d, 1H, J=8.05 Hz, H-1), 4.37 (d, 1H, J=8.30 Hz, H-1), 2.67 (d, 1H, J=2.2
Hz, OH), 2.49 (dd, 1H, J=5.64, 13.3 Hz, H-3"eq), 2.16, 2.03, 2.02, 1.96,
1.88 (5s, 4OAc and NHAc), 1.85 (dd, 1H, J=11.3, 12.9 Hz, H-3"ax), 1.26 (m,
2H, CH.sub.2 Si), 0.05 (s, 9H, SiMe.sub.3).
B) Methyl
3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-1-thio-.beta.-D-galactopyranoside
(20)
A mixture of
1,3,4,6-tetra-O-acetyl-2-deoxy-2-phthalimido-.beta.-D-galactose (680 mg,
1.425 mmol), methylthiotrimethylsilane (0.8 ml, 5.64 mmol), and trimethyl
silyl triflate (0.32 ml, 1.66 mmol) in CH.sub.2 Cl.sub.2 was stirred at
room temperature for 2 days. Diisopropylamine (.about.1 ml) was added,
diluted with CH.sub.2 Cl.sub.2 (.about.50 ml) and washed with saturated
NaHCO.sub.3 solution and water, respectively. The organic layer was dried
(Na.sub.2 SO.sub.4) and evaporated to dryness. Column chromatography
(heptane-EtOAc) gave pure 20 (630 mg, 95%) as an amorphous powder, having
[.alpha.].sub.D.sup.24 +25.degree. (c1, CHCl.sub.3).
.sup.1 H-NMR data (CDCl.sub.3) .delta. 7.87-7.74 (m, 4H, aromatic), 5.87
(dd, 1H, J=3.4, 11.0 Hz, H-3), 5.52 (d, 1H, J=3.2 Hz, H-4), 5.35 (d, 1H,
J=10.6 Hz, H-1), 4.63 (t, 1H, J=10.8 Hz, H-2), 4.25-4.10 (m, 3H, H-6,
H-5), 2.20 (s, 3H, SCH.sub.3), 2.19, 2-05, 1.85 (3s, 9H, 3 OAc).
C) Compound 21
Compound 19 (200 mg, 0,161 mmol), 20 (150 mg, 0.322 mmol) and molecular
sieves 3 .ANG. (0.1 g) in CH.sub.2 Cl.sub.2 --CH.sub.3 CN (2:1, 3 ml) was
stirred under N.sub.2 for 1 h. Silver triflate (0,084 g, 0.327 mmol) was
added, flashed with N.sub.2, cooled to -78.degree. C. Methylsulfenyl
bromide (0.041 g, 0,322 mmol, in 1,2-dichloroethane 0.09 ml) was injected
in 4 portions. The temperature was raised to -28.degree. C. and the
mixture was stirred for 3 h. Diisopropyl amine (0.2 ml) was added and
stirred for 30 min. The mixture was filtered through celite, diluted with
CH.sub.2 Cl.sub.2 (50 ml), and washed with saturated NaHCO.sub.3 solution
and water respectively. The organic layer was dried (Na.sub.2 -SO.sub.4)
and evaporated to dryness. Column chromatography (toluene-EtOH
40:1.fwdarw.30:1) gave 21 (137 mg; 51%) as a powder, having
[.alpha.].sub.D.sup.22 -18.degree. (c 0.7, CHCl.sub.3).
.sup.1 H-NMR data (CDCl.sub.3) .delta. 7.8-7.2 (m, aromatic), 5.81 (d, 1H,
J=8.4 Hz, H-1'"), 5.53 (dd, 1H, J=3.2, 12.4 Hz, H-3'"), 5.41 (m, 1H,
H-4"), 5.36 (bd, J=2.7 Hz, H-4'"), 5.31 (m, 2H, H-7", 8'"), 5.19 (d, 1H,
J=10.3 Hz, NH), 4.56 (dd, 1H, J=8.4, 11.5 Hz, H-2'"), 3.07 (dd, 1H, J=5.7,
9.1 Hz, H-3"eq), 2.18-1.81 (7s, 24H, 7 OAc and NHAc), 1.00 (m, 2H,
CH.sub.2 Si), 0.02 (s, 9H, SiMe.sub.3).
D) TMSEt GM.sub.2 -lactam (22)
Compound 21 (90 mg, 0.054 mmol) and 10% Pd-C(50 mg) in glacial acetic acid
(3 ml) was stirred under hydrogen overnight at room temperature, filtered
through celite and evaporated. The residue was taken up in ethanol (3 ml),
hydrazine hydrate (0.3 ml) was added and stirred at 85 C. for 1 h 20 min.
after which it was diluted to 20 ml (ethanol), evaporated and
co-evaporated with ethanol (5.times.10 ml). The residue was stirred in
pyridine/Ac.sub.2 O ) (1:1, 3 ml) for 1 hour at room temperature and then
evaporated and co-evaporated with toluene (5.times.5 ml). Deacetylation
was done with methanolic NaOMe (0.05M, 2 ml) for 2 hours at room
temperature. The mixture was then decationised with Duolite H.sup.+
resin, filtered, and evaporated. Column chromatography (CHCl.sub.3
-MeOH-H.sub.2 O, 10:5:1) of the residue gave 22 (24 mg, 48.2%) having
[.alpha.].sub.D.sup.22 -29.degree. (c 0.8, MeOH).
.sup.1 H-NMR data (D.sub.2 O) .delta. 4.68 (d, 1H, J=8.41 Hz, H-1'), 4.62
(d, 1H, J=8.1 Hz, H-1'"), 4.47 (d, 1H, J=8.05 Hz, H-1), 4.36-4.26 (m, 2H,
H-4'", H-5"), 3.55 (bd, 1H, J=9.8 Hz, H-7"), 3.42 (dd, 1H, J=8.2, 10.1 Hz,
H-2'"), 3.25 (t, 1H, J=8.5 Hz, H-2), 2.63 (dd, 1H, J=5.95, 14.9 Hz,
H-3"eq), 2.12 (dd, 1H, J=5.13, 14.9 Hz, H-3"ax), 2.03, 2.02 (2s, 6H,
2NHAc), 0.99 (m, 2H, CH.sub.2 Si), 0.00 (s, 3H, SiMe.sub.3).
Example 3
Preparation of TMSEt GM.sub.4 -lactam (29)
A) 2-(Trimethylsilyl)ethyl
3,4,6-tri-O-acetyl-2-azido-2-deoxy-.beta.-D-galactopyranoside (23)
3,4,6-Tri-O-acetyl-2-azido-2-deoxy-.alpha.-D-galactopyranosyl bromide (6 g,
15.2 mmol), 2-(trimethylsilyl)-ethanol (2.7 g, 22.8 mmol) and powdered
molecular sieves 4 .ANG. (14.6 g) in dry CH.sub.2 Cl.sub.2 (100 ml) was
stirred under N.sub.2 for 1 h. Silver silicate (369) was added. After 20
min. stirring, the reaction mixture was filtered through celite and the
filtrate was evaporated to dryness. Column chromatography (heptane-EtOAc
3:1) of the residue gave 23 (5.11 g, 78%) as a syrup.
[.alpha.].sub.D.sup.25 -18.degree. (c 1, CHCl.sub.3).
.sup.1 H-NMR data (CDCl.sub.3) .delta. 5.32 (bd, 1H, H-4), 4.77 (dd, 1H,
J=3.3, 10.9 Hz, H-3), 4.37 (d, 1H, J=8.0 Hz, H-1), 4.22-3.98 (m, 3H, H-6,
OCH.sub.2), 3.85 (m, 1H, H-5), 3.70-3.61 (m, 2H, H-2, OCH.sub.2),
2.15-2.04 (3s, 9H, 30Ac), 1.06 (m, 2H, CH.sub.2 Si), 0.04 (s, 9H,
SiMe.sub.3).
B) 2-(Trimethylsilyl)ethyl 2-azido-2-deoxy-.beta.-D-galactopyranoside (24)
Compound 23 (4.88 g, 11.3 mmol) was stirred in methanolic NaOMe (0.04M, 50
ml) for 90 min. The mixture was decationised with Amberlite
IR-120(H.sup.+) resin, filtered and evaporated to dryness to give 24 (3.24
g, 94%), [.alpha.].sub.D.sup.25 +8.5.degree. (c 0.8, MeOH), m.p.
158.degree.-161.degree. C. (ether-heptane).
.sup.1 H-NMR data (CD.sub.3 OD) .delta. 4.29-4.26 (m, 1H, H-1), 4.10-4.01
(m, 1H, OCH.sub.2), 3.99 (dd, 1H, 1.1, 2.6 Hz, H-4), 3,73 (m, 2H, H-2,3),
3.65 (m, 1H, OCH.sub.2), 3.46 (m, 1H, H-5), 3.43 (m, 2H, H-6), 1.00 (m,
2H, CH.sub.2 Si), 0.05 (S, 9H, SiMe.sub.3).
c) 2-(Trimethylsilyl)ethyl
2-azido-2-deoxy-3,4-isopropylidene-.beta.-D-galactopyranoside (25)
Compound 24 (1.0 g, 3.27 mmol) in 2,2-dimethoxypropane (15 ml) was stirred
in the presence of a catalytic amount (.about.10 mg) of p-toluenesulfonic
acid for 24 h. Triethylamine (.about.0,5 ml) was added and the mixture was
evaporated to dryness and co-evaporated with toluene (2.times.10 ml) to
remove traces of Et.sub.3 N. The residue was refluxed in 80% methanol for
5 h, then concentrated. Column chromatography (heptane-EtOAC. 2:1
containing 0.1% Et.sub.3 N) gave 25 (0.98 g, 86%), [.alpha.].sub.D.sup.25
+40.4.degree. (c 0.9, CHCl.sub.3), m.p. 82.degree.-83.degree. C.
(heptane).
.sup.1 H-NMR data (CDCl.sub.3) .delta. 4.24 (d, 1H, J=8.51 Hz, H-1), 4.10
(dd, 1H, J=2.0, 5.4 Hz, H-4) 4.04-3-81 (m, 6H), 3.62 (m, 1H, OCH.sub.2
CH.sub.2), 3.38 (t, 1H, H-2), 2.07 (dd, 1H, OH), 1.54, 1.34 (2s, 6H,
CMe.sub.2), 1.05 (m, 2H, CH.sub.2 Si), 0.04 (s, 9H, SiMe.sub.3).
D) 2-(Trimethylsilyl)ethyl
2-azido-2-O-benzoyl-2-deoxy-3,4-isopropylidene-.beta.-D-galactopyranoside
(26)
Benzoyl chloride (0.4 g, 3.29 mmol) was added to a solution of 25 (0.87 g,
2.53 mmol) in pyridine (10 ml) at 0.degree. C. After 1 h, water (0.5 ml)
was added. The mixture was diluted with CH.sub.2 Cl.sub.2 (50 ml) and
washed with saturated NaHCO.sub.3 solution and water respectively. The
organic layer was dried (Na.sub.2 SO.sub.4) and evaporated. Column
chromatography of the residue (heptane-EtOAc) gave 26 (1.12 g, 98%) as a
syrup, [.alpha.].sub.D.sup.25 +61.degree. (c 0.9, CHCl.sub.3).
.sup.1 H-NMR data (CDCl.sub.3) .delta. 8.06-7.57 (m, 5H, aromatic), 4.25
(d, 1H, J=8.5 Hz, H-1), 4,17 (dd, 1H, J=2.2, 5.3 Hz, H-4), 3.42 (t, 1H,
H-2), 1.57, 1.36 (2s, 6H, CMe.sub.2), 1.04 (m, 2H, CH.sub.2 Si), -0.01 (s,
9H, SiMe.sub.3).
E) 2-(Trimethylsilyl)ethyl
2-azido-6-O-benzoyl-2-deoxy-.beta.-D-galactopyranoside (27)
Compound 26 (1.07 g, 2.37 mmol) is 80% aqueous acetic acid (15 ml) was
stirred at 90.degree. C. for 2 h. The mixture was concentrated and the
residue was chromatographed (heptane-EtOAc 2:1) to give 27 (0.8 g, 83%),
m.p. 55.degree.-57.degree. C. (ether-heptane), [.alpha.].sub.D.sup.25
37.8.degree. (c 1, CHCl.sub.3).
.sup.1 H-NMR data (CDCl.sub.3) .delta. (8.03-7.45 ml, 5H, aromatic), 4.69
(dd, 1H, J=6.9, 11.4 Hz, H-6), 4.50 (dd, 1H, J=6.5, 11.4 Hz, H-6), 4.32
(d, 1H, J=7.6 Hz, H-1), 4.00 (m, 1H, OCH.sub.2 CH.sub.2, 3.92 (bd, 1H,
H-4), 3.77 (m, 1H, H-5), 3.63 (m, 1H, OCH.sub.2 CH.sub.2), 3.54 (m, 1H,
H-29, 3.42 (dd, 1H, J=3.3, 10.0 Hz, H-3), 1.05 (m, 2H, CH.sub.2 Si), 0.00
(s, 9H, SiMe.sub.3).
F) 2-(Trimethylsilyl)ethyl O-(methyl
5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-.alpha.-D-galacto
-2-nonulpyranosylonate)-(2.fwdarw.3)-O-4-O-acetyl-2-azido-2-deoxy-6-O-benzo
yl-.beta.-D-galactopyranoside (28)
Compound 27 (0.5 g, 1.22 mmol), 10 (0.9 g, 1.51 mmol) and powdered
molecular sieves (3 .ANG., 1.5 g,) in CH.sub.2 Cl.sub.2 --CH.sub.3 CN
(2:3, 40 ml) was stirred under N.sub.2 at room temperature for 1 h. Silver
triflate (0.41 g, 1.61 mmol) was added and the mixture was cooled to
-78.degree. C. Methylsulfenyl bromide (0.19 g, 1.47 mmol) in
1,2-dichloroethane (0.37 ml) was added in 3 portions stirring was
continued at -78.degree. C. for 3 hours 30 min. Diisopropylamine (0.27 ml)
was added and stirred for 30 min. at -78.degree. C. After usual work-up
(see work up procedure for preparation of compound 11), the residue was
chromatographed (MTBE-EtOH 20:1) to give a mixture which was acetylated
(pyridine/Ac.sub.2 O 1:1, 20 ml, 2 h). Solvents were removed and the
residue was chromatographed (EtOAc-toluene 1:1.fwdarw.8:1) to give 28 (661
mg, 58%) as a powder, [.alpha.].sub.D.sup.25 -45.degree. (c 1,
CHCl.sub.3).
.sup.1 H-NMR data (CDCl.sub.3) .delta. 8.02-7.42 (m, 5H, aromatic), 5.61
(m, 1H, H-8'), 5.25 (dd, 1H, J=1.9, 9.3 Hz, H-7'), 5.06 (d, 1H, J=9.96 Hz,
NHAc), 4.99 (m, 2H, H-4, H-4'), 4.62 (dd, 1H, J=3.4, 10.1 Hz, H-3), 4.41
(m, 1H, H-6a), 4.37 (d, 1H, J=8.1 Hz, H-1), 4.31 (dd, 1H, J=2.5, 12.7 Hz,
H-9'a), 4.24 (dd, 1H, J=6.2, 11.3 Hz, H-6b), 4.10 (dd, 1H, J=4.8, 12.7 Hz,
H-9'b), 3.99 (m, 3H), 3.80 (s, 3H, OCH.sub.3), 3.62 (m, 2H), 2.64 (dd, 1H,
J=4.6, 12.5 Hz, H-3'eq), 2.13-2.04 (5s, 15 H, 5 OAc), 1.94 (m, 1H,
H-3'ax), 1.88 (s, 3H, NHAc), 1.08 (m, 2H, CH.sub.2 Si), 0.00 (s, 9H,
SiMe.sub.3).
G) TMSEt GM.sub.4 -lactam (29)
NaBH.sub.4 (204 mg, 5.4 mmol) in ethanol (12 ml) was added dropwise to a
stirred solution of compound 28 (50 mg, 0.0534 mmol), NiCl.sub.2, 6H.sub.2
O (400 mg, 1.68 mmol), H.sub.3 BO.sub.3 (120 mg, 1.0 mmol) in ethanol (5
ml) at 0.degree. C. during 4 h. The mixture was then evaporated and the
residue was taken up in CH.sub.2 Cl.sub.2 (60 ml) and washed with
saturated solution of NaHCO.sub.3 and water, respectively. The organic
layer was dried (Na.sub.2 -SO.sub.4) and evaporated. The residue was
stirred in methanolic NaOMe (0.05M, 5 ml) for 2 h neutralised with AcOH
and evaporated. The residue was filtered through SiO.sub.2 (CH.sub.2
Cl.sub.2 -MeOH 1:1/(CH.sub.2 Cl.sub.2 -MeOH 1:1). The filtrate was
evaporated and stirred with pyridine (2 ml), triethylamine (2 ml) and
4-dimethylaminopyridine (6 mg) for 2 days at room temperature. The
solvents were removed by evaporation and the residue was chromatographed
(CH.sub.2 Cl.sub.2 -MeOH 6:1) to give 29 (18 mg; 61%) as a white powder.
[.alpha.].sub.D.sup.25-.sub.1 3.4.degree. (c 1, H.sub.2 O).
.sup.1 H-NMR data (D.sub.2 O) .delta. 4.57 (d, 1H, J=8.2 Hz, H-1), 4.32 (m,
1H, H-4'), 4.06 (m, 1H, OCH.sub.2 CH.sub.2), 4.02 (bd, 1H, H-4), 3.96 (dd,
1H, J=2.6, 10:7 Hz, H-3), 3.87 (t, 1H, J=10.3 Hz, H-5), 2.55 (dd, 1H,
J=5.4, 10.2 Hz, H-3'eq), 2.01 (s, 3H, NHAC), 1.66 (dd, 1H, J=11.5, 12.9
Hz, H-3'ax), 1.02 (m, 2H, CA.sub.2 Si), 0.00 (s, 9H, SiMe.sub.3).
Example 4
Conformational Studies
Initially, studies were made of the conformational structure of GM.sub.3
-lactone, i.e. the lactone of the naturally occurring GM.sub.3
ganglioside. It was surprisingly found that in contrast to the structure
suggested by Yu et al. cited above, GM.sub.3 -lactone is better described
by two structures where the lactone ring is present as boat-like
conformations (see Scheme 1 below).
##STR11##
The evidence for boat-like conformations of the lactone ring rests on two
observations:
i) the NMR signal of H-4 in the sialic acid residue of the GM.sub.3
-lactone is shifted downfield by .about.0.6 ppm as compared to the parent
GM.sub.3 -ganglioside. Such strong deshielding effects are indicative of a
close contact in space (H . . . O-distance <2.7 .ANG.) between the
hydrogen atom in question and an oxygen atom (Bock, K; Kihlberg, J.;
Magnusson, G. Carbohydr. Res. 1988, 176, 253). In the two boat-like
conformations (Scheme 1) the distance between H-4 of the sialic acid
residue and the carbonyl oxygen is .about.2.5 .ANG., whereas this distance
is considerably longer (.about.3.0 .ANG.) in the chair-like conformation;
ii) molecular mechanics calculations (see U. Burkert and N. L. Allinger
Molecular Mechanics, Am. Chem. Soc. USA, 1982) with GM.sub.3 -lactone
showed that a boat-like conformation is considerably more stable than a
chair-like conformation. Thus, even when the latter was used as the
starting conformation in the calculations, the boat-like conformation was
obtained after energy minimization.
The molecular construction and energy minimization calculations were
carried out on a Apple Macintosh personal computer using the MacMimic/MM2
(91) software package (from InStar Software, Ideon Research Park, S-22370
Lund, Sweden) with the dielectric constant set at 80.
Similar calculations for the corresponding lactam (the bovine serum albumin
conjugate of which was prepared in Example 1 as compound 18) also resulted
in the boat-like conformation. Furthermore, superimposition and
RMS-fitting (by means of the above-described software) of the low-energy
boat conformations of GM.sub.3 -lactone and GM.sub.3 -lactam using all the
ring atoms of the corresponding methyl glycosides showed them to have very
similar over-all shapes with the RMS error being as low as 0.097 .ANG..
This relationship is depicted on the drawing where FIG. 1 shows the
energy-minimized structure of GM.sub.3 -lactone methyl glycoside, and FIG.
2 shows the energy-minimized structure of GM.sub.3 -lactam methyl
glycoside. FIG. 3 is a superimposition of FIGS. 1 and 2 and clearly
illustrates the very close similarity in structure between on the one hand
the natural but hydrolytically unstable ganglioside lactone and on the
other hand the analogous synthetic and hydrolytically stable ganglioside
lactam according to the invention which indicates that the two compounds
are potentially able to exert the same biological activity. For example,
GM.sub.3 -lactam, coupled to bovine serum albumin (Compound 18 in Example
1), should induce an immune response similar to that of the GM.sub.3
-lactone antigen (see Nores et al. cited above), and antibodies should
cross-react with the respective antigens. These lactams should be stable
against hydrolytic cleavage in vivo and therefore keep up a high
concentration of antigen.
Example 5
Immunization Studies
Materials and Methods
Establishment of Monoclonal Antibodies.
The method of Kohler was followed (Immunol. Meth. II (1981), 285-298). The
GM.sub.3 -lactam-BSA conjugate (18, 50 .mu.g) was dissolved in
phosphate-buffered saline (PBS, pH 7.2, 500 .mu.l ), mixed with 500 .mu.l
of Freund's complete adjuvant (FCA, Sigma Chemical Co., St. Louis, Mo.,
USA) and injected subcutaneously (s.c.) into a Balb/c mouse. The
immunization, now using the antigen with Freunds incomplete adjuvant
(FIA), was repeated three times (s.c.) with one week intervals. After 20
days, the mouse received a final intravenous (i.v.) booster dose (50 .mu.g
of 18 in 100 .mu.l of PBS) and seven days later it was splenectomized. The
spleen cells were fused with Sp2/0 myeloma cells at the ratio 1:4 and
hybridomas were screened for reactivity with coated GM.sub.3 -lactam-BSA
(18, 3 .mu.g/ml) in
ELISA (see below).
A synthetic glucose-derived BSA conjugate (40, prepared from glucose
pentaacetate in a manner analogous with the preparation of 18 above),
corresponding to the inner part of 18, and BSA were used as negative
controls. Cells from positive wells were expanded and recloned. More than
300 monoclonal hybridomas were established. Eight of the hybridomas were
chosen at random and their specificities were investigated.
Tissue Culture.
Hybridoma cell lines were maintained in RPMI 1640 medium supplemented with
5% fetal calf serum, 100IU/ml penicillin and 1:50 dilution of
hypoxanthin-thymidine (H.T.) supplement. All tissue culture media and
supplements were from Gibco Ltd., Paisley, Scotland.
Specificity Testing by Binding to Neoglycoproteins and Glycolipids (Table
1).
GM.sub.3 -lactam-BSA (18), Glc-BSA (40), BSA, GM.sub.3 -ganglioside (41;
obtained from Biocarb, Lund, Sweden) and GM.sub.3 -ganglioside lactone
(42) (Yu, R. K., Koerner, T. A. W., Ando, S., Yohe, H. C. and Prestegaard,
J. H. (1985) J. Blochem. 98, 1367-1373) were coated on microtiter plates
using the method described by Nores et al. (J. Immunol. 139 (1987),
3171-3176). All compounds were used at concentrations of 3 .mu.g/ml.
Antibodies (100 .mu.l of supernatant) were added to each of the coated
wells and the amount of bound antibody was detected in ELISA as described
below.
ELISA Screening.
ELISAs were performed by first binding the neoglycoproteins 18 and 40, and
BSA (3 .mu.g/ml in NaHCO.sub.3 -buffer, pH 9.6, 100 Ml ), and the
gangliosides 41 and 42 (6 .mu.g/ml in CH.sub.3 OH, 50 .mu.l) to ELISA
microplates (Costar, Cambridge, Mass, USA). The plates were left over
night at 21.degree. C. in a humified atmosphere (neoglycoprotein plates),
or in a ventilated hood (gangliosides) and blocked with a BSA solution (1%
in PBS buffer, 200 .mu.l/well) for 30 min. The plates were washed with
3.times.200 .mu.l of washing buffer (PBS-0.05% Tween 20). Hybridoma
supernatant (100 .mu.l) was added to each well, the plates were incubated
for 2 h at 21.degree. C., and washed with 3.times.200 .mu.l of washing
buffer. Rabbit antimouse-Ig/alkaline phosphatase-conjugate (Dako A/S,
Glostrup, Denmark) in PBS containing 0.1% BSA was added to each well and
the plates were incubated for 1 h at 21.degree. C., and washed as above.
The phosphatase substrate (p-nitrophenyl phosphate, 5 mg, tablet, Sigma
Diagnostics, St. Louis, Mo., USA) was dissolved in 10 ml of substrate
buffer (pH 9.8; 97 ml diethanolamine and 101 mg MgCl.sub.2
.multidot.6H.sub.2 O , dissolved in H.sub.2 O to a volume of 1000 ml) and
added to the wells (200 .mu.l/well). The plates were incubated for 30 min.
at 37.degree. C. and the optical density (OD) was measured at 405 nm with
a Titertek Multiscan photometer (Flow Labs Ltd., Ayshire, Scotland). Sera
from immunized and pre-immunized Balb/c mice were used as positive and
negative controls, respectively.
Specificity Testing by Inhibition (FIG. 4).
The following 2-(trimethylsilyl)ethyl glycosides were used in the
inhibition studies: GM.sub.3 -TMSEt (43), GM.sub.3 -lactam-TMSEt (12),
GM.sub.2 -lactam-TMSEt (22) GM.sub.4 -lactam-TMSEt (29), Gb.sub.3 -TMSEt
(44) (Kihlberg, J., Hultgren, S. J., Normrk, S. and Magnusson, G. (1989)
J. Am. Chem. Soc. 111, 6364-6368), and asialo-GM.sub.2 -TMSEt (45) as well
as asialo-GM.sub.1 -TMSEt (Ray, A. K. and Magnusson, G. (1992) Acta Chem.
Scand. 46, 487-491).
##STR12##
Each of the glycosides was dissolved in PBS buffer with 0.1% BSA to give a
2 mM solution, which was sequentially diluted with 5 volumes of PBS buffer
in glass tubes. An aliquot (160 .mu.L) of each saccharide solution was
added to aliquots of the antibody solution (160 .mu.l of supernatant), the
plates were incubated over night at 4.degree. C., and tested in ELISA
(FIG. 4).
Isotyping of Monoclonal Antibodies.
Ig class and subclass testing of hybridomas was performed with a commercial
dipstick kit (Holland biotechnology, Aj Leiden, The Netherlands).
Results and Discussion
By immunization of mice with GM.sub.3 -lactam-BSA (18), followed by
establishment of hybridomas, a high number (>300) of antigen-specific
clones were found. The majority of these clones recognized the
sialyl-lactam-galactose portion of the antigen; clones recognizing the
Glc-BSA structure (40) or BSA were not processed further. Considering the
low immunogenicity of gangliosides in general, it is of special interest
to note the high IgG response to GM.sub.3 -lactam, indicating its highly
"non-self" structure and immunogenicity.
Eight randomly chosen hybridomas produced antibodies that were found to
belong to the IgG, .kappa. class (Table 1). They all recognized the
antigen GM.sub.3 -lactam-BSA (18) coated on microtiter plates but did not
bind to BSA, Glc-BSA (40), or GM.sub.3 -ganglioside lactone (42), thus
meeting the objective to use a saccharide analog (18) for immunization and
to obtain antibodies recognizing the natural counterpart (42).
TABLE 1
______________________________________
Binding specificity and subclass of eight randomly chosen
monoclonal antibodies obtained by immunization with GM.sub.3 -
lactam-BSA (18).
Antibody 18 42 41 40 BSA Subclass
______________________________________
P2-D10-H4-H9 (P2-1)
+++.sup.a
-.sup.c
- - - IgG.sub.2b, .kappa.
P2-E12-C9-F9 (P2-2)
+++ - - - - IgG.sub.2b, .kappa.
P3-A12-H4-F3 (P3)
+++ - - - - IgG.sub.2b, .kappa.
P4-C7-H9 (P4-1)
+++ - - - - IgG.sub.2b, .kappa.
P5-F12-D8-B2 (P5-1)
+++ ++.sup.b
- - - IgG.sub.1, .kappa.
P5-F12-E4-D4 (P5-2)
+++ - - - - IgG.sub.1, .kappa.
P5-F12-G5-E6 (P5-3)
+++ ++ - - - IgG.sub.1, .kappa.
______________________________________
.sup.a +++, strong binding, ELISA optical density reading >1.5 at 405 nm.
.sup.b ++, moderate binding, ELISA optical density reading .about.1 at 40
nm.
.sup.c -, no binding.
The hybridomas P3, P5-1, and P5-3 were deposited on 25 June, 1992 in
accordance with the provisions of The Budapest Treaty, with the European
Collection of Animal Cell Cultures, Public Health Laboratory Service,
Centre for Applied Microbiology and Research, Porton Down, United Kingdom,
under the accession numbers 92062591, 92062592, and 92062593,
respectively.
The ability of various compounds to inhibit the binding of the antibodies
produced by the three deposited hybridomas identified above to the antigen
(18) against which the antibodies were raised was tested as a function of
concentration of inhibitor. FIG. 4A shows inhibition curves for antibody
P3 as a function of the concentration of 12 (.smallcircle.), 22
(.quadrature.), 29 (.multidot.) and 43, 44, 45, and 46 (x). FIG. 4B shows
inhibition curves for antibody P5-1 as a function of the concentration of
12 (.smallcircle.), 29 (.quadrature.), and 43, 22, 44, 45, and 46
(.multidot.). FIG. 4C. shows inhibition curves for antibody P5-3 as a
function of the concentration of 12 (.smallcircle.), 29 (.quadrature.),
and 43, 22, 44, 45, and 46 (.multidot.). FIG. 4D shows the ability of the
antigen in dissolved form to inhibit the binding of antibody P3
(.smallcircle.), P5-1 (.quadrature.), and P5-3 (.multidot.) to the antigen
immobilized on the test plates as a function of the concentration of the
dissolved antigen.
As it will be seen from FIG. 4 a-d, the binding of antibody P5-1 and P5-3
to coated 18 was inhibited by soluble 18 and by the lactams 12 and 29,
whereas the glycosides 43, 22 44, 45 and 46 were inefficient as
inhibitors. The fact that GM.sub.2 -lactam-TMSEt (22) was inactive
indicates that the antibody recognizes the epitope of lactamized (and
lactonized) GM.sub.3 -saccharides which in the GM.sub.2 -lactam carries a
sterically hindering GalNAc residue. Binding of antibody P3 (which did not
recognize GM.sub.3 -ganglioside lactone 42) to coated 18 was inhibited by
the lactams 12, 22, and 29. Obviously, the GalNAc residue of 22 did not
hinder the binding. Thus, antibodies P3 and P5-1/P5-3 seem to recognize
different saccharide epitopes. Since antibodies P5-1 and P5-3 recognize
GM.sub.3 -ganglioside lactone (42) but not the open form of GM.sub.3
-ganglioside (41) (Table 1), they are potentially useful for selective
immunohistological detection of GM.sub.3 -ganglioside lactone (42).
Of principal importance is the fact that these antibodies recognize both
the natural GM.sub.3 -lactone as well as the synthetic GM.sub.3 -lactam,
which further confirms that the saccharide conformations are very similar
when bound by the antibodies. Therefore, stable ganglioside lactams may be
generally useful substitutes for unstable ganglioside lactones in active
immunization against ganglioside-expressing tumors and in other biomedical
investigations.
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